Thyroid hormone receptors (TRs) are ligand-dependent, transcriptional regulators of metabolism. TRs repress gene expression in the absence of hormone, which is paradigmatic for other nuclear receptors (NRs) that function as repressors in the unliganded state. Repression is mediated by interaction with corepressors N- CoR (Nuclear Receptor Corepressor) and SMRT (Silencing Mediator of Retinoid and Thyroid receptors), which exist in stoichiometric association with the chromatin-modifiying enzyme, histone deacetylase 3 (HDAC3). HDAC3, in turn, derives its catalytic activity from interacting with N-CoR/SMRT via their unique Deacetylase Activation Domain (DAD). The DAD-dependent N-CoR/SMRT7HDAC3 complex is critical for repression by TR and other NRs in vitro, but the role of this interaction in vivo is unknown. Here we propose to use state of the art methods of gene targeting and mouse phenotyping to test, for the first time, the physiological relevance of the N-CoR7HDAC3 and SMRT7HDAC3 corepressor complexes. We hypothesize that these DAD-dependent interactions are very important, and affect distinct physiological pathways involving NRs. Specific Aim 1 is to determine the physiological function of the N-CoR DAD domain. Knockout of N-CoR is embryonic lethal; we hypothesize that interaction with HDAC3 subserves a subset of N-CoR's developmental and physiological functions. To discover what those functions are, we have generated mice with a point mutation in the N-CoR DAD domain that prevents HDAC3 interaction. Preliminary data demonstrate that mice homozygous for this mutation are viable, with intriguing abnormalities that point to the biological importance of N-CoR7HDAC3. Specific Aim 2 is to determine the physiological function of the SMRT DAD domain. Similar to N-CoR, the physiological role of SMRT7HDAC3 is unknown. We hypothesize that SMRT subserves unique functions that are mediated by HDAC3, and may also have HDAC3-dependent functions that are redundant with those of N-CoR. We will test these hypotheses by generating knockin mice with a point mutation in the SMRT DAD domain. The N-CoR and SMRT homozygous mutant mice, and doubly homozygous mutants, will be carefully analyzed to determine the physiological function of the N-CoR/SMRT7HDAC3 interaction. Specific Aim 3 is to determine the physiological, tissue-specific functions of HDAC3. HDAC3 will be deleted in mice to test the hypothesis that losses of HDAC3 function will phenocopy the doubly homozygous N-CoR/SMRT DAD mutant mice. The HDAC3 knockout will be conditional, enabling us to investigate tissue-specific functions of HDAC3. Together, these innovative and unique studies will elucidate mechanisms regulating transcription repression by TR and other NRs in a physiological context. The insights gained from this work will shed new light on the transcriptional and epigenetic control of key biological pathways, including metabolism and inflammation. This has the potential to lead to new and deeper insights into metabolic disorders, such as obesity, diabetes, and cardiovascular disease, as well as cancer. Relevance: In the past decade, corepressors have emerged as critical regulators of hormone receptors. The proposed studies will innovatively and uniquely elucidate mechanisms regulating the action of hormones and other metabolic regulators. The insights gained from this work will shed new light on key biological pathways, with the potential to lead to new and deeper insights into metabolic disorders, including obesity, diabetes, and cardiovascular disease, as well as cancer.